It is clear that, during phagocytosis by PMN leukocytes, an increased cyanide-insensitive oxygen consumption and an increased metabolism of glucose via the HMP pathway occur simultaneously. Evidence favors the activation of a pyridine nucleotide-dependent oxidase as the initiating event. The specificity of the oxidase for pyridine nucleotide, the cellular location of the oxidase, and the relationship of its activation to control of the HMP pathway remain to be elucidated. A method has been developed to investigate the fate of NADH3 and NADPH3 during phagocytosis by human PMN leukocytes. The approach is designed to make it possible to verify the specificity of the oxidase which is activated during phagocytosis and to confirm the enzyme sequence which regulates HMP pathway activity during phagocytosis. Utilizing a newly described method for determination of hydrogen peroxide generation by leukocyte homogenates and subcellular fractions, the location of the activated oxidase will be investigated and its characteristics evaluated. The importance of the pronounced oxidative metabolism in bactericidal function of PMN leukocytes was indicated by our previous demonstration of the bactericidal defect and associated failure to activate the oxidative metabolic events in PMN leukocytes from patients with chronic granulomatous disease. The continuing comparison of normal PMN leukocytes with PMN from these patients will both contribute to our understanding of the basis of the abnormality and clarify the essential features of the normal response.